Immediately after transcription of a gene begins, the newly synthesized RNA begins to undergo a series of postranscriptional modifications of its structure. These modifications, collectively referred to as RNA processing, include several specific reactions for each of the three major classes of cytoplasmic RNAs - messenger, ribosomal and transfer (mRNA, rRNA and tRNA, respectively) - and seem to be necessary for the production and transport of functional RNA molecules. This proposal focuses upon methylation, one of those modifications which occurs on the precursors to all three types of cytoplasmic RNA. Of particular interest, however, is the role of pre-mRNA methylation upon the other processing events which occur: capping, polyadenylation and splicing. The research proposes to assess the role of methylation by attempting to block methylation in vivo and subsequently analysing the RNA to determine if the other processing events have been inhibited, delayed or unaffected. These research objectives should contribute to our knowledge of how gene expression is regulated, which is central to our understanding of genetic diseases, developmental disorders and cancers, as well as to the application of gene therapy in clinical situations. Cells are cultured under conditions which inhibit methylation, and labeled RNA is purified for analysis. Amount and size of the newly synthesized RNA is determined by gradient sedimentation and dual labeling. Detailed analysis of methylation and cap patterns is possible by enzymatic degradation of the RNA prior to separation of the digest on high performance liquid chromatography (HPLC). Comparisons between the RNAs from inhibited and control cultures should enable evaluation of relationship(s) which exist between methylation and other RNA processing events.